The invention relates to automated and integrated instrumentation and methods useful for the performance of analytical biological assays and reactions, particularly automated screening of low volume samples for new medicines, agrochemicals, cosmetics, and foodstuffs in assays in which multiple effects are simultaneously monitored on gene expression, signal pathway transduction, metabolic networks, and other complex biological systems.
Screening is an important early step in the drug discovery process. Modern biological research requires many diverse assays for specific biological activities in cells or isolated biochemicals in order to discover new biological targets for disease, new medicines directed to those targets, or other chemicals useful in the agrochemical, foodstuffs, cosmetics and other industries. An increasing number of assay types are being developed, including analyzing expression of particular genes, monitoring of activation or inhibition of signal transduction pathways, determining enzyme activity, measuring ion channel activity, quantifying levels of metabolites, and other analysis of biological activities and functions. As new assays become developed, they are put to use in discovering the molecular networks involved in normal physiological regulation of cells, control of gene expression, pathogenesis of various diseases, host physiological responses to pathogenic assault, and other knowledge useful in the diagnosis and treatment of disease. Drug development is beginning to require determination of the effects of combinations of chemical compounds on hundreds of genes and scores of signal transduction pathways to enable design of therapies tailored to the pathogenic mechanisms of a particular disease.
Biological assays often require extensive repetitively performed laboratory procedures to prepare a large number of samples that can then be run in parallel. In drug discovery, for example, a large number of identical assay samples are prepared, and then each sample receives a different, unique chemical compound to determine whether any of those compounds exert an effect on the biological activity probed by the assay that might merit pursuit as a therapeutic. Procedures used to construct these screening assays are generally typical of assay construction. Such procedures and tasks include handling a wide variety of liquids such as chemical reagents, cells, enzymes, substrates, cofactors, buffers, signal development reagent systems, or growth media in accurate and precise quantities. Assay construction is followed by accurate measurement of a signal, which is a change in a measurable physical output, that is produced by the assay and that indicates the time course or extent of the biological activity for which the assay was devised. The performance of assay construction and measurement is sometimes termed “staging”.
Complexity of assay staging has lead to the development of automated laboratory systems including high-throughput assay systems (cf. U.S. Pat. Nos. 5,139,744; 5,985,214; 6,429,025; the contents of which are incorporated by reference in their entirety), which are automated and integrated instruments that handle the various aspects of assay staging. For example, to be efficient, chemical compound screens need to be conducted in a format that can maximize the information about chemical compound of potential interest with regard to a particular target while offering the ability to assess different targets to which the compound may exert its effect. This requires that each compound in libraries of millions of compounds be tested in a wide variety of assays, and each type of assay needs to be replicated as many times as needed to test all compounds.
Similar needs arise in molecular biological assays in which it is desired to understand gene expression. In gene expression analysis, messenger RNA is isolated from cells treated with a particular chemical compound, reverse transcribed to cDNA, amplified by the polymerase chain reaction (PCR) and then hybridized to single-stranded DNA segments encoding particular genes of interest to determine which gene's expression levels were altered by the chemical. Although these procedures can be routinely performed manually when less than 100 genes activities are assayed for each chemical tested, the number of genes of interest may run into the tens of thousands, and the number of chemicals that may exert desired but as yet unknown effects on their expression may number in the billions. The need for automation and increased throughput has lead to the development of automated thermal PCR cyclers and nucleic acid microarrays. In microarrays, robotic liquid dispensers are used to place nanoliter-volume liquid solutions of DNA segments, which encode genes, on glass or plastic substrates as small pads with length dimensions on the order of tens of microns. In an alternate method to liquid spotting, small, 20-nucleotide fragments of the genes are synthesized photolithographically on the substrate. Therefore, instrumentation systems that stage different biological assays and increase the throughput of each assay in a single organized system of work stations will benefit the performance of modern biological research and development.
Storage and assay plates traditionally have 96 or 384 wells. Over a thousand 96-well plates are needed to store 100,000 compounds and equally as many are needed each time a screen is run. A thousand plates can be cumbersome; handling them requires a room- sized automated storage-and-retrieval system. Also, since each well in the 96-well plate must be filled with about 200 microliters of reagent, the system needs to have a supply of about 20 liters to run a screen of 100,000 compounds. Screening in 96-well plates is quite a large operation.
Large, complex automation systems like those required to screen low-density plates must be located in a centralized facility. Only experts can operate the equipment, so companies that do screening usually have a dedicated screening team. An assay is transferred from the scientist who develops it to the screening team where it may sit in a queue for months. Running the screen may also take months depending on the number of compounds and the complexity of the assay.
To meet the increased demands and needs for assay performance, considerable attention has been devoted to decreasing the sample volume of assays while improving the ability of assays to discriminate small changes in biological activity, a process termed “miniaturization”. Miniaturization efforts have been advanced with the intention of devising automated and integrated workstations capable of constructing and measuring numerous assays in parallel to achieve high throughput of experimental results. For example, the microtiter plate or multiwell platform has evolved from an industry-standard format of 96 wells, each containing a volume of ˜200 μL, to increasingly higher densities of 384, 1536, 3456, and larger numbers of wells. This was achieved by subdividing the 8×12 well standard format of the 96-well plate to enable the use of sample volumes of less than 50, 10, and 5 μL to accommodate the demands of assay miniaturization to down to and the need to achieve high throughput handling of assays in parallel. About thirty 3456-well microplates are enough to screen 100,000 compounds, and required reagent volume drops by a factor of 100 compared to a 96-well plate. Principles and implementation of assay plate well miniaturization for high-throughput screening are disclosed in U.S. Pat. No. 6,232,114, the contents of which are incorporated by reference in their entirety. These platforms are now mass-produced standards in the industry, and available from numerous suppliers including Greiner, Coming, Nunc, and others. Multiwell microtiter plates provide a basis for much of the automated assay staging in high-throughput screening.
Miniaturization and parallelization efforts have also lead to the development of liquid handling and spectrometric measurement instrumentation specially suited to automated high-throughput biology. Automated liquid handling is required to achieve the throughput requirements in miniaturized assays because the manually operated piston-plunger dispensers used for pipetting volumes >10 μL become cumbersome, inaccurate, and imprecise when repetitively transferring smaller volumes. Miniaturization of automated liquid handling has encompassed handling at least two ranges of volumes needed for miniaturized assays. The first range is from 1 microliter to 10 nanoliters, which encompasses the volume increments needed to construct assays from a variety of constituents comprising cells, media, assay signal development reagents, enzymes or other assay reagents, in which the total volume per well is less than 5 μL. A widely used technology is the solenoid-actuated valve, in which the liquid to be dispensed is maintained at a constant hydrostatic pressure behind the valve, and the valve solenoid is actuated for a few milliseconds to dispense the liquid through an outlet with an orifice diameter of about 100 μm. To dispense the same liquid to multiple miniaturized wells in an assay platform simultaneously, multiple dispensers are aligned with their orifices spaced according to the well pitch using a fixturing mechanism such as a multi-orifice plate. The target multiwell plate is automatically repositioned under the orifice or multiple orifice fixture at locations that are integral multiples of the center-to-center spacing of the wells to fill the wells of the plate. This type of positioning achieves accuracy and reliability because the different tubes and lines that feed the dispenser are not subject to movement and compression and, hence, ejection pressure variance. Dispensing systems of this type are disclosed in U.S. Pat. No. 5,985,214, the contents of which are incorporated by reference in their entirety. Nonetheless, in some systems, the dispenser head is moved over a stationary receptacle. This type of dispenser is available commercially from several vendors, such as Genomic Solutions, Biodot, and others, in different configurations adaptable to the dimensions of the platform in which the assays are constructed.
The second range of volumes needed in miniaturized assays extends below 10 nL. The requirement for this volume range arises in assays where relatively small volumes of a chemical compound concentrate are added to an assay during construction. The need for the small relative volume arises because the compound may be dissolved in a non-aqueous solvent, such as dimethylsulfoxide or benzene, which may exert its own effect in a biological assay. The objective is to dilute the small volume of solvent (e.g., 1 nL) with the relatively much larger volume of aqueous assay diluent (1 μL) to a concentration where biological effects are mitigated. This situation arises in the screening of chemical compound libraries for new therapeutics. To obtain these low volumes, ink-jetting technologies such as thermal- or piezo-actuation have been adapted to biological assay construction. A commercially available piezo-actuated dispenser for miniaturized assay construction is the Microdrop from PE Biosystems. A new technique for small-volume dispensing is surface acoustic wave control in which the surface of the liquid to be dispensed is energized to produce a standing stationary wave. Energization is provided by a small acoustic lens, such as a curved piezoelectric ceramic lens brought into contact with the bottom of the container of the liquid. Dispensing of pico- or nano-liter sized drops is actuated by the addition of a high-amplitude transient pulse to the energizing wave, which causes reorganization standing wave modes into a jet that projects from the liquid surface and coalesces into a drop the volume of which depends on the amplitude of the actuation pulse. This technique is disclosed in U.S. Pat. No. 4,751,530, the contents of which are incorporated by reference in their entirety, and is available as a commercial system from EDC Biosciences as the HTS-01. Thus, a variety of technologies for liquid handling are presently available for integration into high-throughput biological screening systems.
Rapid detection and measurement of the signals developed in miniaturized biological assays has required several innovations. One advance has been the invention of multiwell assay platforms with clear well bottoms having high transmittance to the wavelengths of light generated by the biological assay. In addition, plastic, injection-moldable materials have been developed to provide a composition for both the clear well bottoms and black well sidewalls materials with greatly decreased intrinsic fluorescence (autofluorescence) at the wavelengths of light used to excite the fluorescent assay signal development systems used to detect and measure biological activity. These advances in plate technology greatly decrease both the background fluorescence originating from the plate materials as well as extraneous fluorescence originating from assays in neighboring well of the platform and allow the small fluorescence signals developed by a small number of cells in a single well to be accurately measured. These inventions are disclosed in U.S. Pat. No. 6,517,781, the contents of which are incorporated by reference in their entirety.
Miniaturized optical assemblies or heads enable spectrometric signals to be accurately and precisely measured in miniaturized assay wells as disclosed in U.S. Pat. No. 5,985,214, the contents of which are incorporated by reference in their entirety. The clear bottom of a single well may provide a window diameter of only 0.9 mm or less, and the assay signal may originate from as few as 300 fluorophores in only 10 to 20 cells in the well. Thus, these optical heads need to be accurately positioned to within only a few tenths of a millimeter away from the well bottom in order to enable injecting the excitation light into the assay well being measured without appreciable introduction of excitation light into adjacent wells. In addition, the heads need to provide sufficient numerical aperture to enable capture enough of the resulting light emitted by the assay in the well to allow an appreciable signal to be measured that is proportional to the biological activity being probed in the assay. This has lead to the development of optical heads using a ball lens that is positioned close to the well bottom and that is interposed between the clear well bottom and the face of a bundle of optical fibers. This fiber bundle is subdivided into two or more sub-bundles so that one sub-bundle conveys the light of the excitation wavelengths from a source, such as an arc lamp, through an interference filter or other means to isolate the excitation wavelengths of light to the ball lens for illumination of the well contents through the clear bottom. The ball lens captures a portion of the light emitted by the signal development system of the assay in the well through the clear well bottom and focuses this light onto the face of the optical fiber bundle. The remaining sub-bundles transmit this light away from the ball lens. At the opposite end of each sub-bundle is an interference filter that passes only the wavelengths of emitted light that are to be measured in the assay to the face of a photosensitive detector such as a photodiode, photomultiplier tube or charge-coupled device. The multiplicity of sub-bundles enables multiple emission wavelengths to be measured from multiple fluorophores in the assay, such as donor and acceptor emission intensities of resonance energy transfer probes, or different fluorophores in the same cell that report the activities of different biological molecules.
To read the assay samples in the different wells of a multiwell plate, the optical head is brought up to its action position under the clear bottom of each well. For stability reasons, this is typically achieved by repositioning the plate over a fixed optical head by the use of automated X-Y positioners. Because the plate bottom may not be sufficiently flat to within the ˜100 μm spacing requirements of the optical head with respect to the well bottom to achieve a constant numerical aperture for reading of each well, topographical corrections have been implemented. The vertical position of the center of each well bottom is measured, such as by a laser displacement sensor, and then the vertical location of each well is used to position the plate vertically over the optical head to obtain the same numerical aperture for every well. Since modern photodetectors can be operated in single-photon detection mode, only a very brief dwell time of the well over the optical head is needed to obtain a spectrometric reading. This enables, for example, a multiwell plate consisting of an array of 48×72 wells (3456 wells total) to be read in as little as 2 min. Thus, spectrometric readers are available that are capable of handling the throughput and sensitivity demands posed by running a large number of miniaturized biological assays in parallel.
As the components needed to perform the various tasks of assay construction and measurement have been developed, integration of these devices into automated systems has become the key to enabling high-throughput biological assays. One of the earliest efforts at an automated laboratory workstation is the Biomek (Beckman Instruments, Fullerton, Calif.) as disclosed in U.S. Pat. No. 5,139,744, the contents of which are incorporated by reference in their entirety. In the Biomek, multiwell plates are located at different areas on a work surface that are functionally defined so as to identify each plate as being either a source of an assay reagent material or a destination for the mixing of an assay. The functionally defined locations provide the sets of coordinates needed by the controller for a robotic arm to position the arm at specific locations over each plate. An array of micropipettes connected to a positive-displacement pump was fixed at the end of the robotic arm. By using the plate positions coded by the functionally defined work surface, the controller could position the micropipettes in the wells of the source plates, aspirate the chemical reagent samples needed, raise the arm, position the pipettes in the wells of the destination plates, and dispense the reagents to the assay under construction. Assay construction could be programmed so as to aspirate and dispense liquid volumes selected by a user from a variety of source and destination plates. The Biomek automated the many repetitive volumetric liquid transfer tasks involved in assay construction and could be adapted to a wide variety of solution-based assays. More extensive assay task control and integration of several laboratory work stations performing different assay tasks is embodied in the system for automated drug discovery screening of chemical compound libraries disclosed in U.S. Pat. No. 5,985,214, the contents of which are incorporated by reference in their entirety. In their ultra-high throughput screening system, a robotic arm was used to place multiwell plates containing compounds from the compound library onto a set of parallel conveyer lanes, which queued the plates into plate stackers that provided plates to different liquid handling and assay measurement modules located at spur transports, perpendicular to the main transport lanes. For example, one liquid handler aspirated the chemicals from the compound library plate, transferred each chemical to a separate well in a fresh plate, and then added a diluent to each well to provide the dilution of chemical in the assay buffer necessary for performance of the assay. These dilution plates were then maintained in a plate stacker and delivered one at a time to a transport lane where they were conveyed to a different workstation where the diluted chemicals were transferred to plates in which the assays were constructed.
Despite its advantages, miniaturization is still not feasible for most companies. One hurdle is that new instrumentation is required to access the small wells of high-density microplates because the dispensers and plate readers designed for low-density plates will not work with 1536- or 3456-well microplates. Anther issues is that most of the instruments available today cannot transfer liquid from one high-density plate to another. This means that compounds must still be stored in thousands of low-density plates inside a room-sized machine. Even if screening is being done in high-density microplates, the advantages of miniaturization are only half realized.